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a Seminis Vegetable Seeds, Inc., 37437 State Highway 16, Woodland, CA 95695
b Dep. of Agronomy, Univ. of Wisconsin, Madison, WI 53706; former address for T.C. Osborn, Dep. of Agronomy, Univ. of Wisconsin, Madison, WI 53706
* Corresponding author (Tom.Osborn{at}seminis.com).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONConclusionsREFERENCES |
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Abbreviations: DH, doubled haploid • NIL, near-isogenic line • QTL, quantitative trait loci
Received for publication April 9, 2007.
a Seminis Vegetable Seeds, Inc., 37437 State Highway 16, Woodland, CA 95695
b Dep. of Agronomy, Univ. of Wisconsin, Madison, WI 53706; former address for T.C. Osborn, Dep. of Agronomy, Univ. of Wisconsin, Madison, WI 53706
* Corresponding author (Tom.Osborn{at}seminis.com).
Wild relatives and unadapted forms of crop plants are important reservoirs of allelic variation that can be accessed through wide crosses for improvement of crop plants. In this paper, we describe insights and innovations from the use of wide crosses to transfer favorable alleles from wild or unadapted forms of two crops: canola (oilseed Brassica napus L.) and tomato (Lycopersicon esculentum Mill.). The canola research involves the use of unadapted forms (winter types and resynthesized B. napus) in wide crosses to spring-seeded types to increase seed yield of hybrids. The tomato research involves the use of wild tomato relatives to improve several traits, including disease resistance and fruit quality. These two crop plants provide a broad range of insights and innovations that have been gained from wide crosses in plant breeding research, including the impact of chromosomal rearrangements on allelic variation among unadapted forms of B. napus, and innovations that can be achieved from stacking elite alleles in tomato. The examples presented in this paper highlight the value of using DNA markers for understanding and applying the results of wide crosses.
Abbreviations: DH, doubled haploid • NIL, near-isogenic line • QTL, quantitative trait loci
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONConclusionsREFERENCES |
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The level of success and ultimate value of transferring useful genetic variation through wide crosses depends on many factors. One involves the breadth of diversity that can be accessed to introduce useful variation. A second is the speed and efficiency with which useful alleles can be transferred to meet the demands of a changing world. A third factor is whether useful variation can be transferred from wild and unadapted sources without the deleterious traits that also exist within these sources. Backcross breeding is quite effective in eliminating deleterious traits that are conferred by genes unlinked to the desired genes, but linkage of desired and undesired genes can present a critical obstacle that must be overcome for success. A fourth factor is how broadly a particular useful allele can be utilized in different genetic backgrounds. Alleles that are broadly useful (elite alleles) are obviously more valuable than alleles that confer desirable phenotypes in only a limited number of genetic backgrounds.
While all of these factors have biological constraints that may limit success, technologies have been developed that can have a large impact on the value of transferring novel genetic variation through wide crosses. Techniques involving embryo rescue and tissue culture are quite useful for recovering progeny from wide crosses, and these techniques have extended the range of variation that is accessible through wide crosses. DNA marker technologies also have been valuable as tools for identifying, tracking, and determining the effects of alleles from wild species and unadapted sources. Thus, DNA markers can improve the speed and efficiency of developing superior cultivars that contain novel traits from these sources.
In this article, we present some insights and innovations derived from efforts to introgress useful genetic variation from wild and unadapted sources into elite cultivated germplasm. We do this by reviewing the results of research on two crop plants, canola (oilseed Brassica napus L.) and tomato (Lycopersicon esculentum Mill.). This seemingly odd pairing of species was chosen based mainly on the authors having had some experience in each. The canola examples come from research conducted by two of the authors (Osborn and Kramer) at the University of Wisconsin, and the tomato examples come from published literature and the experiences of three of the authors (Osborn, Graham, and Braun) at Seminis Vegetable Seeds. Since these two crop species represent very different biological systems and since the goals and applications of wide crosses have been very different within each, they provide a broad range of insights and innovations that have been gained from wide crosses in plant breeding research.
Introgression of Alleles from Unadapted Sources in Canola
Sources of Diversity in Canola
Oilseed B. napus (called canola or double low rapeseed when it is low in eruic acid and gluosinolates) has been bred as two very different forms adapted to two different cultivation environments. Spring or annual forms are seeded in the spring and flower and set seed in the summer; and winter or biennial forms are seeded in the fall and overwinter requiring vernalization to flower and set seed the following spring and summer. These two forms have distinct gene pools based on DNA marker analysis (Diers and Osborn, 1994), and pedigree records indicate that breeders have made very few crosses between the pools, except to transfer the genes for canola quality from spring to winter forms by backcrossing (L. Sernyk, personal communication, 2003). Although winter forms are unadapted to spring cultivation, and vice versa, the winter forms represent a source of allelic variation not present in the spring germplasm that could be transferred to the spring types through wide crosses. Allelic variation of winter types also could be expanded by introgressing alleles from spring types. An expanded gene pool may be useful for improvement of many traits in oilseed B. napus. Since canola cultivars are now sold largely as hybrids, increased allelic diversity among parents of hybrids could contribute to increased heterosis and seed yield of hybrid cultivars.
Another potential source of new alleles for oilseed B. napus is from the two diploid progenitor species. Brassica napus (n = 19) is an amphidiploid species that originated from hybridization of the diploid species (or close relatives of these) B. rapa (n = 10) and B. oleracea (n = 9). These diploid species contain much more phenotypic and genetic diversity than B. napus. Brassica rapa includes vegetable forms, such as turnip, Chinese cabbage, and pak choi, along with oilseed and wild forms; and B. oleracea includes vegetable forms, such as cabbage, broccoli, cauliflower, kales, and others, along with wild forms. The diploid species can be hybridized and chromosomes doubled to resynthesize B. napus that is fully cross-fertile with natural B. napus (Song et al., 1993; U, 1935). Thus, the diploid species also represent a source of allelic diversity that could be transferred into B. napus by wide crosses, and they may contain alleles that when introgressed would increase the heterosis and seed yield of hybrids.
Effects of Introgression on Seed Yield of Spring Hybrids
Our initial phenotypic analysis of several introgression lines derived one winter cultivar, Major, suggested that this source could provide favorable alleles for seed yield in a spring hybrid background (Butruille et al., 1999a). To assess additional unadapted sources and to identify the genomic regions affecting seed yield, we created and analyzed larger segregating populations of spring types containing genomic introgressions from Major, additional winter sources, and a resynthesized B. napus. The first set of these populations were created as inbred backcross lines using the German winter cultivar Ceres as a donor backcrossed to two spring parents (Butruille et al., 1999b). A second set of populations were created as F1–derived doubled haploid (DH) lines using two winter cultivars as donor sources (Major and Samourai) after converting to spring growth habit by crossing or backcrossing (Quijada et al., 2004b) and using a Chinese cultivar (Huadbl2, intermediate winter–spring type) and a resynthesized spring type (Resyn) directly as donor parents (Udall et al., 2004) in crosses to a spring parent. The advantage of using populations of F1–derived DH lines is that they have much more power for detection of quantitative trait loci (QTL) effects than do backcross-derived populations (Kaeppler, 1997). The populations were evaluated as lines per se and as testcross progenies in two to four environments for seed yield and other agronomic traits (Butruille et al., 1999b; Quijada et al., 2004b; Udall et al., 2004). DNA markers were used to identify genomic regions affecting the measured traits (Butruille et al., 1999b; Quijada et al., 2006; Udall et al., 2005, 2006).
Our main objective was to determine if these diverse sources could contribute alleles for increased seed yield in spring hybrids, and the details of results from analyses of the discovery populations has been reported elsewhere (Butruille et al., 1999b; Quijada et al., 2006; Udall et al., 2006). We anticipated that these unadapted sources would contribute some negative effects on seed yield of hybrids, and we observed negative effects, most of which occurred at loci in the B. oleracea portion of the B. napus genome (N11–N19, Fig. 1 ). However, positive effects also were observed from introgression of each of these sources, and most of the positive effects occurred at loci in the B. rapa portion of the genome (N1–N10, Fig. 1). Several other agronomic traits, in addition to seed yield, were recorded for these test cross progenies, and data were analyzed to identify QTL controlling these traits. The locations of some QTL for other traits, such as days to flowering and disease resistance, coincided with some QTL for seed yield, suggesting possible causes for these seed yield effects; however, there were no consistent correlations between seed yield and these other traits across all environments and loci (Butruille et al., 1999b; Quijada et al., 2006; Udall et al., 2006).
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Another important question is whether alleles having positive effects in one genetic background will have positive effects when transferred to different hybrid genetic backgrounds. We previously tested this for the N3 and N14 QTL alleles from the cultivar Ceres by crossing these segments into three different male parents and then comparing the donor and recurrent parent alleles as test cross progenies with two different testers. The experimental details and results have been published elsewhere (Quijada et al., 2004a) and are summarized in Fig. 1. We found that the N14 segment from Ceres had no significant effect on seed yield in any of the new genetic backgrounds; and thus, the effect observed in the original discovery population may have been specific to the genetic background or environments tested, or an experimental artifact. For the N3 introgression segment from Ceres, we found variable effects on seed yield compared to the recurrent parent segments, either positive, negative, or neutral, depending on the male parent and tester background. We also have conducted an initial test of other donor segments in different genetic backgrounds and we observed variable results depending on the genetic background and environment (Kramer, 2007). These results indicate that for complex quantitative traits, such as seed yield, alleles from unadapted sources that appear to be favorable in one genetic background may not be elite in all backgrounds. Thus, extensive testing in different genetic backgrounds and environments may be required to determine the value of these alleles, and for hybrid crops both male parent and tester genetic background must be considered.
Potential Impact of Homoeologous Transpositions
Our analyses of B. napus populations derived from wide crosses suggest that some QTL effects may be due to segregation of chromosomal rearrangements. As described earlier, the B. napus genome consists of two subgenomes derived from the diploid progenitors, and these subgenomes have a high degree of homeology. Although most meiotic chromosome pairing occurs between homologous chromosomes, homoeologous chromosomes (or chromosome segments) from the two subgenomes pair and recombine at a high frequency, as indicated by the frequent occurrence of homoeologous chromosome transpositions (Sharpe et al., 1995; Udall et al., 2005). These transpositions result in segments of the B. rapa genome replacing homoeologous segments of the B. oleracea genome, and visa versa, and they can exist either as reciprocal or nonreciprocal transpositions.
The parents of our QTL discovery populations contained several different homoeologous chromosome transpositions that segregated in the populations and were associated with QTL variation (Osborn et al., 2003; Quijada et al., 2006; Udall et al., 2005, 2006). In three genomic regions segregating for rearrangements (N11, N13, and N16, Fig. 2 ), the unadapted donor parents contained transpositions resulting in segments that were homologous to the corresponding segment on the homoeologous chromosome. The presence of these transpositions increased intersubgenome homozygosity, and they also were associated with reduced seed yield in DH lines and/or testcross progenies. In another genome region segregating for a rearrangement (N10, Fig. 2), the four unadapted donor parents contained nonrearranged segments and the spring parent had a homoeologous chromosomal transposition in the corresponding segment. The presence of the nonrearranged segment increased intersubgenome heterozygosity. Surprisingly, these segments were associated with reduced seed yield of DH lines, but this was probably because they also contained an allele for susceptibility to a bacterial disease that mapped as a QTL in the same genomic region. However, the tester contained an allele for resistance, and in test cross progenies, the donor segment was associated with increased seed yield.
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Introgression of Alleles from Wild Species in Tomato
Sources and Traits
The cultivated tomato (Lycopersicon esculentum, syn. Solanum lycopersicum L.) has several closely related wild species, which, together with tomato, form the small genus Lycopersicon. To resolve systematic issues, Lycopersicon has recently been reclassified in the genus Solanum. Recognizing the extensive body of literature that uses the familiar Lycopersicon names, including most of the references cited herein, Lycopersicon nomenclature will be used in this article.
Selection and breeding to adapt tomato to specific growing conditions and uses has been on-going for over 200 yr. Before 1925, improvements were largely accomplished by selection within heterogeneous cultivars or selection for spontaneous mutants. Beginning in the late 1920s, selection within segregating populations allowed more rapid cultivar improvement programs (Boswell, 1937; Stevens and Rick, 1986). The narrow genetic base of cultivated tomato forced breeder and plant pathologists to look to closely related wild species for allelic variation; and thus, the wild relatives of tomato have been used extensively as sources of alleles to improve cultivated tomato germplasm (Table 1 ).
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Most improvements from the use of wide crosses in tomato have been to transfer pathogen resistance genes (Table 1). The impetus for using wild relatives to improve disease resistance in tomato has been attributed to Weber and Ramsey (1926) and Alexander et al. (1942). One of the first disease resistance alleles to be transferred from a wild relative was for resistance to the soil-borne pathogen Verticillium dahliae Kleb. Although Verticillium resistance was identified in 1932 in L. peruvianum, it was not until 1952 that resistant cultivars were released (Stevens and Rick, 1986). This timeline would likely be shorter today with the use of DNA marker technologies.
In some cases the introgression of alleles for disease resistance from wild species has had a tremendous impact on tomato cultivation. One example is the Mi-1 allele, which was introgressed into tomato from L. peruvianum (Smith, 1944) and confers resistance to nematodes. This allele has been introduced into many tomato cultivars and it allows cultivation in nematode-infested soils throughout the world without the need for costly chemical fumigation. Another example is the Ty-1 allele, which was introgressed from L. chilense, and confers resistance to Tomato yellow leaf curl virus (Hoogstraten, 2002; Zamir et al., 1994). This virus has become a serious threat to tomato cultivation in most tropical and subtropical areas of the world, and the inclusion of Ty-1 in cultivars is becoming essential for continued production in these areas. Nearly every wild species that can be crossed with tomato has contributed alleles for disease resistance, and they will undoubtedly continue to be an important source for resistances to evolving pathogen pressures.
Wild relatives of tomato also are sources of alleles for improving fruit quality. One trait that has received considerable attention is the concentration of soluble solids in tomato fruit measured by brix value (Eshed and Zamir, 1995; Fulton et al., 2002; Osborn et al., 1987). Brix is a component of yield for processed tomato products and contributes to flavor in fresh market tomato. Most of the wild species have been shown to contain alleles that increase brix value when introgressed into tomato. In some cases, the wild donor did not have higher brix than cultivated tomato, although it had alleles that increased brix when introgressed into tomato. This phenomenon, termed "cryptic" genetic variation, can be revealed by gene mapping with DNA markers, and is not unexpected, having been observed in many studies of various crops and traits, including many of the studies cited in this paper. The wild relatives of tomato are also sources of alleles that affect other components of flavor, such as the concentration of specific sugars and organic acids (Fulton et al., 2002), and the accumulation of nutritional compounds, such as lycopene (Bernacchi et al., 1998). Thus, wild species may be an important source of traits that benefit consumers.
There are some limitations in the ability of breeders to select for the phenotypes of many traits that have been introgressed from wild to cultivated tomato. For example, because pathogen transportation is limited by various regulatory agencies, screening for resistance to Tomato yellow leaf curl virus is mostly limited to those areas where the pathogen occurs naturally. In addition, breeding progress is often slowed because the tests can only be performed once a year and pathogen pressure can be variable from year to year. Similarly, selection for nematode resistance, conferred by the Mi-1 allele can be difficult to perform reliably. The use of molecular markers has overcome both of the above limitations, and provides the breeder the ability to perform horticultural evaluations on plants that have not been compromised with exposure to these pathogens.
The measurement of some quantitative traits, such as soluble solids content, is time consuming and the effects of favorable alleles may be small and easily missed. For these and other traits, selection of the allele of interest using linked DNA markers can be valuable because they can be rapidly performed with very low error rates. It is also possible to distinguish homozygotes and heterozygotes, allowing breeders to fix alleles quickly, or to compare the performance of the allele in the homozygous and heterozygous states. Thus, DNA markers can improve the speed and efficiency of transferring alleles from wild sources to cultivars.
Linkage to Deleterious Traits
The wild relatives of tomato contain many traits that are considered deleterious for production or consumption, such as poor plant habit, small fruit, soft fruit, off-color fruit, or low fruit set. Backcrossing is quite effective in eliminating deleterious alleles from wild parents at loci that are unlinked to the target locus, and DNA markers can be used to help speed this process by allowing breeders to select more efficiently for recurrent parent genotype (Hospital et al., 1992). However, Hansen (1959) estimated that even after several generations of backcrossing, the size of donor segments can remain quite large, and large chromosome segments from wild species will often contain deleterious alleles linked in coupling to the desired target alleles. DNA markers allow a means to efficiently select for recombinants within the segment, and analysis of recombinant individuals or their progeny with markers allows researchers to map and separate the linked effects, hopefully recovering plants that have lost the deleterious trait but maintain the target trait. This approach is quite useful if the traits under study are quantitative and require replicated trials to detect. An example of this was provided by Frary et al. (2003), who identified a QTL allele from L. chmielewskii that increased brix value but also was associated with orange fruit, an undesirable trait for many tomato markets. Evidence that the two effects were due to linked loci and that the coupling phase linkage could be broken was obtained through the development and analysis of recombinant near isogenic lines (NILs).
An alternative strategy is to select for alleles at other loci that diminish or negate the deleterious effects of linkage drag. For example, poor fruit color could be overcome by selecting alleles for higher lycopene or other factors that affect color at other loci. This strategy has the disadvantage of requiring manipulation of more than just the favorable target locus to obtain an acceptable cultivar. It also will limit future advancements that can be made because the deleterious alleles will continue to be maintained in the cultivated gene pool by linkage to the desired alleles.
Elite and Nonelite Alleles from Wild Species
An important question is whether the alleles brought in from wild species are superior to all existing alleles in the tomato germplasm at that locus (elite alleles), or if they represent a member of an allelic series that is superior to some but not others (nonelite alleles). Alleles that have been introgressed for qualitative disease resistance have, for the most part, proven to be elite to all existing tomato alleles, and this is anticipated because the resistance was absent from the existing germplasm necessitating the wide cross. The status of alleles for quantitative traits is largely unknown. A possible exception is an invertase allele from L. pennellii, which has a point mutation that affects the gene product's Michaelis constant for the substrate, sucrose (Fridman et al., 2004). However, the effect of this allele in relation to multiple alleles from tomato has not been reported. Cultivated tomato has very limited allelic variation compared to the variation in wild relatives, but it cannot be assumed that a favorable allele discovered by contrasting with one other allele will be elite when compared to all tomato alleles. This must be tested empirically.
DNA markers provide a tool to help identify an allelic series and then test their effects to determine if any behave as elite alleles. Using DNA markers in a tightly defined region around the target locus, different haplotypes can be identified among cultivated germplasm. These haplotypes can be compared phenotypically against each other and against favorable alleles introgressed from wild species. One way to do this is by backcrossing each allele into one or more genetic backgrounds to create NILs and then comparing their phenotypic effects. However, the generation of NILs takes a lot of time and effort, and they may contain different undetected donor segments that could affect the phenotype. An alternative approach is to bulk individuals having segments for the two contrasting alleles from segregating populations and testing the relative trait value of the two bulks. This averages the effects of other segregating alleles in the populations and can be effective if the parents are not too diverse. Additional information can be obtained about potential epistatic and environmental effects by evaluating two alleles in more that one segregating population and in multiple environments.
Stacking Elite Alleles
During the short history of allele introgression from wild relatives of tomato, a large number of elite alleles have been introduced into the cultivated germplasm. These introduced elite alleles, in addition to those already residing within the tomato gene pool, represent a tremendous resource for cultivar improvement. But they also can present a challenge when attempting to combine many elite alleles in marketable cultivars.
The challenge of stacking elite alleles has a practical and simple solution: making commercial hybrid combinations using inbreds, each of which having elite alleles at different loci. This approach will not work for traits conferred by recessive alleles, but for those with dominant, or at least a degree of dominance suitable for trait expression, the use of hybrids to stack elite alleles from the two inbred parents is quite effective. This deployment strategy also can be effective in reducing the effects of linkage drag associated with elite alleles if the drag is conferred by recessive or partially recessive allelic interactions.
A difficulty can arise if a genomic region has more than two loci for which elite alleles are desirable. The strategy of stacking through hybrid deployment can accommodate elite alleles at only two loci in a repulsion phase linkage arrangement, and if an elite allele at a third linked locus is needed and available from another source, simple hybrid deployment is not an option for the three alleles. A solution is to combine alleles at two of the loci in coupling phase linkage. This solution was achieved recently for the Mi-1 and Ty-1 genes (Hoogstraten and Braun, 2005). These two genes are located near the centromere of tomato chromosome 6, along with several other important disease resistance genes that have been introgressed from wild relatives (resistance to Oidium and two races of Cladosporium). While most introgressions from wild species of tomato have some linkage drag, the introgressions containing Mi-1 and Ty-1 each display considerable drag, which is particularly apparent in the homozygous state and under stress conditions. Thus, breeders cannot combine these resistance alleles in hybrid cultivars without also creating a hybrid with poor horticultural performance. Several studies have shown that recombination is suppressed in this genomic region (Kaloshian et al., 1998; Zhong et al., 1999), perhaps as much as 50-fold compared with the rest of the genome, which may be due to an inverted chromosomal segment in susceptible compared to resistant tomato (Seah et al., 2004). With this suppression of recombination, the use of molecular markers is useful in identifying rare recombinants that can be tested for improved performance. Using this approach, the most efficacious resistance alleles from two wild species have been juxtapositioned in cis (Hoogstraten and Braun, 2005). This allows breeders to deliver both of these important alleles in hybrid cultivars from one inbred, while retaining the ability to mask genetic drag with the other hybrid parent. It also allows breeders to deliver additional important traits that are controlled by loci in this region through the use of a second inbred parent containing the additional desired allele in hybrid combinations. It is likely that this type of solution will be required more frequently in the future as more elite alleles are discovered and required in commercial tomato cultivars.
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Conclusions |
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TOPABSTRACTINTRODUCTIONConclusionsREFERENCES |
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Received for publication April 9, 2007.
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TOPABSTRACTINTRODUCTIONConclusionsREFERENCES |
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